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From 2010.igem.org

PCR

Materials:

- Pfx Polymerase (Invitrogen)

- 10x Pfx Bufer (Invitrogen)

- Enhancer (Invitrogen)

- 50 mM MgSO4

- 10 mM dNTPs

- Primer solutions 5 pmol/μL

- Template DNA (plasmid at 50 pg – 1 ng/μL)

Protocol:

First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice. Preparation of reaction mixture:

1. Gently vortex and briefly centrifuge all solutions after thawing

2. Keep solutions on ice

3. Add to a thin walled PCR tube, on ice, for a 100 μL reaction:

1× pre-mix

Component	Sample
Pfx polymerse	0.6 μL
10x Pfx buffer	5 μL
Enhancer	5 μL
10 mM dNTPs	1.5 μL
50 mM MgSO4	1 μL
Primer 1	3 μL
Primer 2	3 μL
DNA template	1 μL
H20	29.9 μL

4. Gently vortex the sample and briefly centrifuge (5 sec) to collect all droplets at the bottom of the tube

5. Place samples from the ice in a thermal cycler preheated to 95 °C and start the previously programmed PCR.

PCR program:

Step	Annealing Temperature	Time, min:sec	Number of cycles
Initial denaturation	x °C *	2:00	1
Annealing	95 °C	2:00	1
Extension	68 °C	1:00	1
Denaturation	95 °C	1:00	25
Annealing	x °C *	1:00	25
Extension	68 °C	1:00	25
Final Extension	68 °C	10:00	1

* Calculate the optimal annealing temperature = 3x G/C + 2x A/T